/ . Biochem., 81, 1505-1509 (1977)
Accumulation of Phosphoenolpyruvate in Red Cells Incubated
with the Phosphate Ester in an Acidified Isotonic
Sucrose Medium1
*Department of Biochemistry, and "Department of Cardiac Surgery,
Kyushu University School of Medicine, Higashi-ku, Fukuoka,
Fukuoka 812
Received for publication, September 2, 1976
Accumulation of exogenous phosphoenolpyruvate against the concentration gradient was observed when human red cells were incubated in an acidified isotonic sucrose medium. Fluoride
increased the apparent accumulation by inhibition of the intracellular metabolic interconversion of the phosphate compound. The accumulation appeared to be specific for phosphoenolpyruvate and the accumulation rate for 3-phosphoglycerate, which has a molecular size
and pKt similar to those of phosphoenolpyruvate, was less than one-tenth of the rate for phosphoenolpyruvate. Red cells incubated in the acidified sucrose medium tended to adhere to
each other when examined with a scanning electron microscope.
Organic phosphate compounds have been generally
regarded as being unable to permeate through the
red cell membrane (/), and this would appear to
be a requirement if the cells are to conserve phosphate compounds within the cells. Similarly,
organic phosphate compounds supplied exogenously are usually considered not to be metabolized
by the cells. Evidence, however, has recently
accumulated for the penetration of nucleotides into
cells
1
This study was supported in part by a research grant
from the Ministry of Education, Science and Culture of
Japan.
1
Present address: Department of Biochemistry, Faculty of Medicine, Kanazawa University, Kanazawa,
Ishikawa 920.
Abbreviations: P-Prv, phosphoenolpyruvate; 3-P-Gly,
3-phosphoglycerate.
Vol. 81, No. 5, 1977
We have previously reported that phosphoenolpyruvate (P-Prv) was accumulated in human
red cells when the cells were incubated in an
acidified isotonic sucrose solution with P-Prv.
The cells showed a normal glycolytic activity when
they were further incubated in neutral Ringer's
medium and the accumulated P-Prv inside the cells
was metabolized to pyruvate, monophosphoglycerates, and 2,3-bisphosphoglycerate (5).
This report deals with the accumulation of
P-Prv in red cells incubated with the phosphate
ester in an acidified isotonic sucrose medium, and
the scanning electron microscopic observation of
these cells. The phosphate ester seems to be unique
among organic phosphate compounds; the accumulation rates of other phosphate esters, e.g. 3phosphoglycerate (3-P-Gly), fructose 1,6-bisphosphate, or ATP, were very much slower than that
of P-Prv under the conditions used.
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Naotaka HAMASAKI,* Akio TOMODA,'. • Hiroaki HARASAKI,**
and Shigeki MINAKAMI*
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N. HAMASAKI, A. TOMODA, H. HARASAKI, and S. MINAKAMI
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Accumulation of P-Prv—When red cells were
incubated at 37°C in the acidified sucrose medium
containing about 10 mM P-Prv, the P-Prv concentration inside the cells increased rapidly to 13.1 mM
in 4 min against the concentration gradient, with a
concomitant decrease of the extracellular P-Prv
concentration to 4.5 mM (Fig. la). The P-Prv
concentration of cells suspended in the acidified
medium without P-Prv was about 10 /*M and it did
not increase on incubation. Little release of hemoglobin, enzymes or phosphate esters was observed
up to 4 min, but hemolysis was observed on further
incubation.
The cells loaded with P-Prv also had high
concentrations of monophosphoglycerates, which
are supposed to be formed from P-Prv inside the
cells. The concentrations of ATP and hexose
monophosphates were essentially unchanged.
Cells incubated for less than 4 min showed an apparently normal glycolytic activity when they were
resuspended in Ringer's solution at pH 7.4 and
incubated at 37°C. The loaded P-Prv was metaThe resuspended samples were deproteinized bolized rapidly to monophosphoglycerates, 2,3with 2 volumes of 0.6 N perchloric acid. All pro- bisphosphoglycerate and pyruvate, as reported
cedures after sampling were done as quickly as previously (5).
NaF and ouabain had no effect on the accumupossible at low temperature to minimize metabolic
lation of P-Prv against the concentration gradient
transformation of the accumulated metabolite.
Analytical Procedures—Metabolites and nu- and this apparent uphill accumulation was not
cleotides were determined enzymatically (6). energy-dependent. The addition of NaF was
Hemoglobin was determined by the method of advantageous during quantitative estimation of the
Drabkin (7). Measurements of the intracellular accumulation to inhibit the metabolic conversion
and extracellular pH were carried out with a Radi- P-Prv to 3-P-Gly and pyruvate and also for the
ometer G297/G2 capillary electrode attached to a prevention of hemolysis. The rate of accumulation
was about 2 /imol/ml of cells per min (Fig. lb).
PHM 64 pH-meter.
The accumulation rate was faster than the
Scanning Electron Microscopy—One drop of
the cell suspension was fixed with 10 ml of 1 % glycolytic rate by several orders of magnitude, and
glutaraldehyde in sucrose medium of corresponding the accumulation was not inhibited by fluoride,
pH (pH 4.5 and 7.4; 420mOsm). The specimens which means that the accumulation of P-Prv was
were post-fixed with 1 % osmium tetroxide in 0.1 M not due to the metabolic formation of P-Prv in the
cacodylate. They were dehydrated with ethanol, cells. Furthermore, phosphorylation of pyruvate
dried by the CO,-critical point drying method and is energetically unlikely and the ATP concentration
observed with a JEOL JSM-2 scanning electron in the cells did not change substantially during the
accumulation. Thus, the accumulation of exomicroscope.
Reagents—All the enzymes, glycolytic inter- genous P-Prv in red cells incubated in an acidified
mediates, and nucleotides used were from C.F. sucrose medium may be ascribed to transport of
/. Biochtm.
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Cell Incubation—Citrated blood stored at 4°C
in ACD (acid-citrate-dextrose) solution was obtained from the Fukuoka Red Cross Blood Center.
Ususally, blood stored for 1-2 days was used. Red
cells were washed thoroughly with an isotonic saline
solution by centnfugation and further washings
with an isotonic sucrose medium (250 mM sucrose,
5 mM KG, 5 mM MgCli, 2 mM potassium phosphate buffer pH 7.4, 10 mM glucose) were carried
out. The washed cells were suspended in the
sucrose medium. The suspension was incubated
in a thermostatted vessel with magnetic stirring at
37°C under a pH stat (Hiranuma PS-11 precision
pH-stat). After preincubation for about lOmin,
organic phosphate esters (converted to the acid
form by treatment with cation exchange resin) were
added; the pH of the suspension was adjusted to
pH 4.5 and kept constant by the addition of 0.1 N
HC1. Samples were removed into 10 volumes of
the cold isotonic sucrose medium at the tunes
specified and centrifuged in a refrigerated centrifuge
at 1,500X0 for 2 min, The precipitate was resuspended in the medium and the hematocrit value
of the red cell suspension was determined.
Bdhringer u Sohne. Other reagents were of analytical reagent grade.
EXOGENOUS PHOSPHOENOLPYRUVATE ACCUMULATION IN RED CELLS
20 f ( b )
20 [ • ( a )
NaF(-)
1507
NaFtO
10
10
Fig. 1 The accumulation of P-Prv inside red cells at pH 4.5. Incubation was
started by the addition of P-Prv with subsequent acidification of the suspension
to pH 4.5 after preincubation for 10 mm at 37°C without (a) or with (b) 10 mM
NaF in the sucrose medium at neutral pH. The pH of the suspension was
adjusted to 4.5 as described in "MATERIALS AND METHODS." Little
hemolysis was observed up to 4 min in incubation with or without NaF, while 9%
hemolysis at 7 min incubation with NaF and 14% hemolysis at 5 mm incubation
without NaF were observed. The hematocnt value of the suspension was about
30% at the beginning of incubation. O, intracellular concentration of P-Prv;
• , intracellular concentration of 3-P-Gly; x , P-Prv concentration of the incubation medium.
TABLE I. The accumulation rate of P-Prv and the differences between intracellular pH (pHi) and extracellular
pH (pH0). Red cells were suspended in isotonic media containing various concentrations of sucrose and KCI. The
hematocnt was 8%. The pH of red cell suspensions was adjusted by the addition of 0.1 N HCI at 37°C. The
supernatant and red cells were separated by centnfugation for 30 s The pHi was measured after hemolyzing the
cells by the addition of 6-12 vols of distilled water. The pHi and pHe were measured at 37°C. The accumulation
rate of P-Prv inside the cells was determined separately in the corresponding medium. Measured values are shown
as mean ± S.E.
Media
pH.
pHi
pH!-pH.
250 mM sucrose
4. 58 ±0.03
5.19±0.04
0.60±0. 07
(«=4)
(«=4)
(«=4)
200 mM sucrose
30 mM KCI
4.61 ±0.05
(n = 5)
4. 92 ±0.05
0.31 ±0.02
(« = 5)
(n = 5)
125 mM sucrose
75 mM KCI
4.6O±0.03
4. 73 ±0.05
0.13±O.O4
(«=4)
(«=4)
(n=4)
87.5 mM sucrose
97.5 mM KC1
4.62±0.06
4. 74±0.08
0.12±0.03
(« = 3)
(« = 3)
(«=3)
Vol. 81, No. 5, 1977
Accumulation rate
of P-Prv
(//mol/ml cells per min)
2.0
1.0
0.8
0.5
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0
3
6
Incubation Time (min)
0
3
6
Incubation Time(mln)
N. HAMASAK1, A. TOMODA, H. HARASAKI, and S. MrNAKAMI
1508
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the phosphate ester into the cells.
Effect of Sucrose Concentration—The rate of
P-Prv accumulation was decreased when sucrose
was partially replaced by KC1. The intracellular
pH of red cells suspended in a sucrose medium was
appreciably higher than that of cells suspended in
an isotonic NaCI or KG solution, as expected from
the Donnan-Gibbs equilibrium (8). The intracellular pH of cells incubated in the acidified
sucrose medium was 0.6 unit higher than the pH
of the medium whereas the difference for cells
incubated in the 87.5 rriM sucrose-97.5 mM KG
medium was 0 12 (Table I). The accumulation rate
decreased from 2.0 to 0.5 //mol/ml of cells per min
in the latter medium (Table I). Further decrease
in the sucrose concentration of the medium might
cause a decrease in the accumulation rate, but could
not be carried out in an isotonic KG medium
because of the fragility of the cells at this pH.
Partial replacement of sucrose with NaCI similarly
reduced the accumulation rate.
This accumulation of the anion against the
concentration gradient may be explained by the
Donnan-Gibbs theory. The intracellular pH of red
cells incubated in the sucrose medium was about
0.6 unit higher than the intracellular pH, which
60
.- 3.0
•a
0
3
6
Incubation Time ( min )
Fig. 2. Comparison of the accumulation rates of
P-Prv and 3-P-Gly. The initial concentrations of P-Prv
and 3-P-Gly in the incubation medium were 4.4 mM and
5.7 mM, respectively. Conditions of incubation were
the same as in Fig. lb. The hematocrit value of the
suspension was about 20% at the beginning of incubation. O, intracellular concentration of P-Prv; • , intracellular concentration of 3-P-Gly.
Fig. 3. Scanning electron micrograph of red cells
(x 1,800). a: cells incubated in the sucrose medium at
pH 7.4, 37°C, for 2 min; b: cells incubated in the sucrose
medium at pH 4.5, 37°C for 2 mm; c: cells incubated in
the sucrose medium, the pH of which was readjusted to
7.4 by the addition of 0.1 N NaOH (see the text).
/. Biochem.
EXOGENOUS PHOSPHOENOLPYRUVATE ACCUMULATION IN RED CELLS
Scanning Electron Microscpoic Observation of
the Cells—Red cells incubated in the isotonic
sucrose medium at pH 4.5 for 2 min at 37°C tended
to adhere to each other and to assemble into
clusters. The constituent cells of the cluster were
spherocytic (Fig. 3a, b). This tendancy may
reflect the decrease in negative charge on the cell
Vol. 81, No. 5, 1977
surface at low pH. When the incubation medium
was readjusted to pH 7.4 by the addition of 0.1 N
NaOH and incubated for a further 4 min, the red
cells still aggregated but the clusters seemed to be
looser. The shapes of the constituent cells of the
clusters returned to the discoid form and were
clearly different from the shapes of the cells at pH
4.5 (Fig. 3b, c).
We thank Dr. Jumchi Tokunaga for advice on scanning
electron microscopy.
REFERENCES
1. Bishop, C. & Surgenor, D.M. (1964) The Red Blood
Cell p. 161, Academic Press, New York
2 Ryan, W.L. & Durick, M.A. (1972) Science 177,
1002-1003
3. Plunkett, W., Lapi, L , Ortiz, P.J., & Cohen, S.S.
(1974) Proc. Natl. Acad. Sci. U.S. 71, 73-77
4. Hochberg, A.A. & Rappoport, S. (1974) Biochem.
Biophys. Res. Commun. 60, 456-459
5. Tomoda, A., Hamasaki, N., & Minakami, S. (1975)
Biochem. Biophys. Res. Commun. 66, 1127-1130
6. Minakami, S., Suzuki, C , Saito, T., & Yoshikawa,
H. (1965) / . Biochem. 58, 543-550
7. Drabkjn, D.L. (1950) /. Biol. Chem. 185, 231-245
8. Lacella, P.L. & Rothstein, A. (1966) / . Gen. Physiol.
50, 171-188
9. Oesper, P. (1951) in Phosphorous Metabolism (McElroy, W.D. & Glass, B., eds.) Vol. 1, pp. 523-536,
Johns Hopkins Baltimore
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may lead to an apparent uphill accumulation of
P-Prv according to the membrane equilibrium
theory, if can permeate through the membrane.
Accumulation of 3-P-Gly—At acidic pH,
negative charges of phosphate compounds and the
membrane are expected to decrease, which may
facilitate transport. If the transport is restricted
•solely by charge and molecular size, we might
expect that other organic phosphate compounds
with similar molecular size and pKx would also
penetrate the membrane.
We used 3-P-Gly to test this possibility. The
molecular weight of the compound is 186 and it
h a s / ^ ' s of 5.68, 3.42, and 1.42 (9), which are quite
similar to those of P-Prv (molecular weight 168,
pK^'s 6.38, 3.5 (9) and probably around 1.5).
When red cells were incubated in the sucrose
medium containing NaF, P-Prv, and 3-P-Gly, the
accumulation rate of 3-P-Gly was less than onetenth of the P-Prv accumulation rate (Fig 2). This
observation suggests that the accumulation of PPrv may be due to the selective nature of the membrane.
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